Transgenic plants with enhanced traits

ABSTRACT

This disclosure provides transgenic plants having enhanced traits such as increased yield, enhanced nitrogen use efficiency and enhanced drought tolerance; propagules, progeny and field crops of such transgenic plants; and methods of making and using such transgenic plants. This disclosure also provides methods of producing hybrid seed from such transgenic plants, growing such seed and selecting progeny plants with enhanced traits. Also disclosed are transgenic plants with altered phenotypes which are useful for screening and selecting transgenic events for the desired enhanced trait.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35USC § 119(e) of U.S.provisional application Ser. No. 61/635,946, filed on Apr. 20, 2012, andis herein incorporated by reference in its entirety.

INCORPORATION OF SEQUENCE LISTING

The sequence listing file named “38-21(58904)0001_SeqListing.txt”, whichis 64,196 bytes (measured in MS-WINDOWS) and was created on Mar. 5,2013, is electronically filed herewith and incorporated herein byreference in its entirety.

FIELD OF THE INVENTION

Disclosed herein are plants having enhanced traits such as increasedyield, increased nitrogen use efficiency and increased water useefficiency; propagules, progenies and field crops of such plants; andmethods of making and using such plants. Also disclosed are methods ofproducing seed from such plants, growing such seed and/or selectingprogeny plants with enhanced traits.

SUMMARY OF THE INVENTION

An aspect of this disclosure provides a plant comprising a recombinantDNA molecule comprising a polynucleotide encoding a polypeptide, whereinthe nucleotide sequence of the polynucleotide is selected from the groupconsisting of: a) a nucleotide sequence set forth as SEQ ID NO: 1, 3, 5,7, 9, 11, 13, 15, 17, or 19; b) a nucleotide sequence encoding a proteinhaving the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16,18, or 20-29; c) a nucleotide sequence with at least 90%, at least 91%,at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99% identity to SEQ ID NO: 1, 3, 5, 7,9, 11, 13, 15, 17, or 19; and d) a nucleotide sequence encoding aprotein with at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99% identity to SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, or20-29; wherein said plant has enhanced trait as compared to a controlplant, and wherein said enhanced trait is selected from the groupconsisting of increased yield, increased nitrogen use efficiency, andincreased water use efficiency.

Another aspect of this disclosure provides a plant comprising arecombinant DNA molecule of the disclosure, wherein said plant is amonocot plant or is a member of the family Poaceae, wheat plant, maizeplant, sweet corn plant, rice plant, wild rice plant, barley plant, rye,millet plant, sorghum plant, sugar cane plant, turfgrass plant, bambooplant, oat plant, brome-grass plant, Miscanthus plant, pampas grassplant, switchgrass (Panicum) plant, and/or teosinte plant, or is amember of the family Alliaceae, onion plant, leek plant, garlic plant;or wherein the plant is a dicot plant or is a member of the familyAmaranthaceae, spinach plant, quinoa plant, a member of the familyAnacardiaceae, mango plant, a member of the family Asteraceae, sunflowerplant, endive plant, lettuce plant, artichoke plant, a member of thefamily Brassicaceae, Arabidopsis thaliana plant, rape plant, oilseedrape plant, broccoli plant, Brussels sprouts plant, cabbage plant,canola plant, cauliflower plant, kohlrabi plant, turnip plant, radishplant, a member of the family Bromeliaceae, pineapple plant, a member ofthe family Caricaceae, papaya plant, a member of the familyChenopodiaceae, beet plant, a member of the family Curcurbitaceae, melonplant, cantaloupe plant, squash plant, watermelon plant, honeydew plant,cucumber plant, pumpkin plant, a member of the family Dioscoreaceae, yamplant, a member of the family Ericaceae, blueberry plant, a member ofthe family Euphorbiaceae, cassava plant, a member of the familyFabaceae, alfalfa plant, clover plant, peanut plant, a member of thefamily Grossulariaceae, currant plant, a member of the familyJuglandaceae, walnut plant, a member of the family Lamiaceae, mintplant, a member of the family Lauraceae, avocado plant, a member of thefamily Leguminosae, soybean plant, bean plant, pea plant, a member ofthe family Malvaceae, cotton plant, a member of the family Marantaceae,arrowroot plant, a member of the family Myrtaceae, guava plant,eucalyptus plant, a member of the family Rosaceae, peach plant, appleplant, cherry plant, plum plant, pear plant, prune plant, blackberryplant, raspberry plant, strawberry plant, a member of the familyRubiaceae, coffee plant, a member of the family Rutaceae, citrus plant,orange plant, lemon plant, grapefruit plant, tangerine plant, a memberof the family Salicaceae, poplar plant, willow plant, a member of thefamily Solanaceae, potato plant, sweet potato plant, tomato plant,Capsicum plant, tobacco plant, tomatillo plant, eggplant plant, Atropabelladona plant, Datura stramonium plant, a member of the familyVitaceae, grape plant, a member of the family Umbelliferae, carrotplant, or a member of the family Musaceae, banana plant; or wherein theplant is a member of the family Pinaceae, cedar plant, fir plant,hemlock plant, larch plant, pine plant, or spruce plant.

Another aspect of this disclosure provides a plant comprising arecombinant DNA molecule of the disclosure, wherein the recombinant DNAmolecule further comprises a promoter that is operably linked to thepolynucleotide encoding a polypeptide, wherein said promoter is selectedfrom the group consisting of a constitutive, inducible, tissue specific,diurnally regulated, tissue enhanced, and cell specific promoter.

Another aspect of this disclosure provides a plant comprising arecombinant DNA molecule of the disclosure, wherein said plant is aprogeny, propagule, or field crop. Such field crop is selected from thegroup consisting of corn, soybean, cotton, canola, rice, barley, oat,wheat, turf grass, alfalfa, sugar beet, sunflower, quinoa and sugarcane.

Another aspect of this disclosure provides a plant comprising arecombinant DNA molecule of the disclosure, wherein said plant is aprogeny, propagule, or field crop. Such propagule is selected from thegroup consisting of a cell, pollen, ovule, flower, embryo, leaf, root,stem, shoot, meristem, grain and seed.

Another aspect of this disclosure provides a method of producing a plantcomprising: introducing into a plant cell a recombinant DNA comprising apolynucleotide encoding a polypeptide, wherein the nucleotide sequenceof the polynucleotide is selected from the group consisting of: a) anucleotide sequence set forth as SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15,17, or 19; b) a nucleotide sequence encoding a protein having the aminoacid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20-29; c)a nucleotide sequence with at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99% identity to SEQ ID NO: 1, 3, 5, 7, 9, 11, 13,15, 17, or 19; and d) a nucleotide sequence encoding a protein with atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%identity to SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20-29; andgrowing a plant from said plant cell.

Another aspect of this disclosure provides a method of producing a plantcomprising: introducing into a plant cell a recombinant DNA molecule ofthe disclosure; growing a plant from said plant cell; and selecting aplant with an enhanced trait selected from increased yield, increasednitrogen use efficiency, and increased water use efficiency as comparedto a control plant.

Another aspect of this disclosure provides a method of increasing yield,increasing nitrogen use efficiency, or increasing water use efficiencyin a plant comprising: producing a plant comprising a recombinant DNA ofthe disclosure wherein said plant has an enhanced trait selected fromthe group consisting of increased yield, increased nitrogen useefficiency, and increased water use efficiency as compared to a controlplant; crossing said plant with itself, a second plant from the sameplant line, a wild type plant, or a second plant from a different lineof plants to produce a seed; growing said seed to produce a plurality ofprogeny plants, and selecting a progeny plant with increased yield,increased nitrogen use efficiency, or increased water use efficiency.

Another aspect of this disclosure provides a plant comprising arecombinant DNA molecule comprising a polynucleotide encoding apolypeptide, wherein the nucleotide sequence of the polynucleotide isselected from the group consisting of: a) a nucleotide sequence setforth as SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, or 19; b) anucleotide sequence encoding a protein having the amino acid sequence ofSEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20-29; c) a nucleotidesequence with at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99% identity to SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, or 19;and d) a nucleotide sequence encoding a protein with at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to SEQ IDNO: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20-29; wherein said plant has atleast one phenotype selected from the group consisting of anthocyanin,biomass, canopy area, chlorophyll score, plant height, water applied,water content and water use efficiency that is altered for said plant ascompared to a control plant.

DETAILED DESCRIPTION OF THE INVENTION

In the attached sequence listing:

SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, and 19 are nucleotidesequences of the coding strand of the DNA molecules used in therecombinant DNA imparting an enhanced trait in plants, each represents acoding sequence for a protein.

SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 are amino acidsequences of the cognate proteins of the DNA molecules with nucleotidesequences 1, 3, 5, 7, 9, 11, 13, 15, 17, and 19.

SEQ ID NOs: 21-29 are amino acid sequences of homologous proteins.

As used herein a “plant” includes whole plant, transgenic plant,meritem, shoot organ/structure (for example, leaf, stem and tuber),root, flower and floral organ/structure (for example, bract, sepal,petal, stamen, carpel, anther and ovule), seed (including embryo,endosperm, and seed coat) and fruit (the mature ovary), plant tissue(for example, vascular tissue, ground tissue, and the like) and cell(for example, guard cell, egg cell, pollen, mesophyll cell, and thelike), and, progeny of same. The classes of plants that can be used inthe disclosed methods are generally as broad as the classes of higherand lower plants amenable to transformation and breeding techniques,including angiosperms (monocotyledonous and dicotyledonous plants),gymnosperms, ferns, horsetails, psilophytes, lycophytes, bryophytes, andmulticellular algae.

As used herein a “transgenic plant” means a plant whose genome has beenaltered by the stable integration of recombinant DNA. A transgenic plantincludes a plant regenerated from an originally-transformed plant celland progeny transgenic plants from later generations or crosses of atransgenic plant.

As used herein a “control plant” means a plant that does not contain therecombinant DNA that imparts an enhanced trait, A control plant is usedto identify and select a transgenic plant that has an enhanced trait. Asuitable control plant can be a non-transgenic plant of the parentalline used to generate a transgenic plant, for example, a wild type plantdevoid of a recombinant DNA. A suitable control plant can also be atransgenic plant that contains the recombinant DNA that imparts othertraits, for example, a transgenic plant having enhanced herbicidetolerance. A suitable control plant can in some cases be a progeny of ahemizygous transgenic plant line that does not contain the recombinantDNA, known as a negative segregant, or a negative isoline.

As used herein a “transgenic plant cell” means a plant cell that istransformed with stably-integrated, recombinant DNA, for example, byAgrobacterium-mediated transformation or by bombardment usingmicroparticles coated with recombinant DNA or by other means. A plantcell of this disclosure can be an originally transformed plant cell thatexists as a microorganism or as a progeny plant cell that is regeneratedinto differentiated tissue, for example, into a transgenic plant withstably-integrated, recombinant DNA, or seed or pollen derived from aprogeny transgenic plant.

As used herein a “propagule” includes all products of meiosis andmitosis, including but not limited to, plant, seed and part of a plantable to propagate a new plant. Propagules include whole plants, cells,pollen, ovules, flowers, embryos, leaves, roots, stems, shoots,meristems, grains or seeds, or any plant part that is capable of growinginto an entire plant. Propagule also includes graft where one portion ofa plant is grafted to another portion of a different plant (even one ofa different species) to create a living organism. Propagule alsoincludes all plants and seeds produced by cloning or by bringingtogether meiotic products, or allowing meiotic products to come togetherto form an embryo or a fertilized egg (naturally or with humanintervention).

As used herein a “progeny” includes any plant, seed, plant cell, and/orregenerable plant part comprising a recombinant DNA of the presentdisclosure derived from an ancestor plant. A progeny can be homozygousor heterozygous for the transgene. Progeny can be grown from seedsproduced by a transgenic plant comprising a recombinant DNA of thepresent disclosure, and/or from seeds produced by a plant fertilizedwith pollen or ovule from a transgenic plant comprising a recombinantDNA of the present disclosure.

As used herein a “trait” is a physiological, morphological, biochemical,or physical characteristic of a plant or particular plant material orcell. In some instances, this characteristic is visible to the humaneye, such as seed or plant size, or can be measured by biochemicaltechniques, such as detecting the protein, starch, certain metabolites,or oil content of seed or leaves, or by observation of a metabolic orphysiological process, for example, by measuring tolerance to waterdeprivation or particular salt or sugar concentrations, or by themeasurement of the expression level of a gene or genes, for example, byemploying Northern analysis, RT-PCR, microarray gene expression assays,or reporter gene expression systems, or by agricultural observationssuch as hyperosmotic stress tolerance or yield. Any technique can beused to measure the amount of, comparative level of, or difference inany selected chemical compound or macromolecule in the transgenicplants, however.

As used herein an “enhanced trait” means a characteristic of atransgenic plant as a result of stable integration and expression of arecombinant DNA in the transgenic plant. Such traits include, but arenot limited to, an enhanced agronomic trait characterized by enhancedplant morphology, physiology, growth and development, yield, nutritionalenhancement, disease or pest resistance, or environmental or chemicaltolerance. In some specific aspects of this disclosure an enhanced traitis selected from the group consisting of drought tolerance, increasedwater use efficiency, cold tolerance, increased nitrogen use efficiency,increased yield, and altered phenotypes as shown in Tables 3-5. Inanother aspect of the disclosure the trait is increased yield undernon-stress conditions or increased yield under environmental stressconditions. Stress conditions can include, for example, drought, shade,fungal disease, viral disease, bacterial disease, insect infestation,nematode infestation, cold temperature exposure, heat exposure, osmoticstress, reduced nitrogen nutrient availability, reduced phosphorusnutrient availability and high plant density. “Yield” can be affected bymany properties including without limitation, plant height, plantbiomass, pod number, pod position on the plant, number of internodes,incidence of pod shatter, grain size, efficiency of nodulation andnitrogen fixation, efficiency of nutrient assimilation, resistance tobiotic and abiotic stress, carbon assimilation, plant architecture,resistance to lodging, percent seed germination, seedling vigor, andjuvenile traits. Yield can also be affected by efficiency of germination(including germination in stressed conditions), growth rate (includinggrowth rate in stressed conditions), ear number, seed number per ear,seed size, composition of seed (starch, oil, protein) andcharacteristics of seed fill.

Also used herein, the term “trait modification” encompasses altering thenaturally occurring trait by producing a detectable difference in acharacteristic in a plant comprising a recombinant DNA encoding apolypeptide of the present disclosure relative to a plant not comprisingthe recombinant DNA, such as a wild-type plant, or a negative segregant.In some cases, the trait modification can be evaluated quantitatively.For example, the trait modification can entail an increase or decrease,in an observed trait as compared to a control plant. It is known thatthere can be natural variations in the modified trait. Therefore, thetrait modification observed entails a change of the normal distributionand magnitude of the trait in the plants as compared to a control plant.

Increased yield of a plant of the present disclosure can be measured ina number of ways, including test weight, seed number per plant, seedweight, seed number per unit area (for example, seeds, or weight ofseeds, per acre), bushels per acre, tons per acre, or kilo per hectare.For example, corn yield can be measured as production of shelled cornkernels per unit of production area, for example in bushels per acre ormetric tons per hectare. This is often also reported on a moistureadjusted basis, for example at 15.5 percent moisture. Increased yieldcan result from improved utilization of key biochemical compounds, suchas nitrogen, phosphorous and carbohydrate, or from improved responses toenvironmental stresses, such as cold, heat, drought, salt, shade, highplant density, and attack by pests or pathogens. This disclosure canalso be used to provide plants with improved growth and development, andultimately increased yield, as the result of modified expression ofplant growth regulators or modification of cell cycle or photosynthesispathways. Also of interest is the generation of plants that demonstrateincreased yield with respect to a seed component that may or may notcorrespond to an increase in overall plant yield.

The present disclosure relates to a plant with improved economicallyimportant characteristics, more specifically increased yield. Morespecifically the present disclosure relates to a plant comprising apolynucleotide of this disclosure that encodes a polypeptide, whereinthe plant has increased yield as compared to a control plant. Manyplants of this disclosure exhibited increased yield as compared to acontrol plant. In an embodiment, a plant of the present disclosureexhibited an improved trait that is a component of yield.

The present disclosure relates to a plant with improved economicallyimportant characteristics, more specifically increased yield. Morespecifically the present disclosure relates to a plant comprising apolynucleotide of this disclosure that encodes a polypeptide, whereinthe plant has increased yield as compared to a control plant. Manyplants of this disclosure exhibited increased yield as compared to acontrol plant. In an embodiment, a plant of the present disclosureexhibited an improved trait that is a component of yield.

Reference herein to an increase in yield-related traits can also betaken to mean an increase in biomass (weight) of one or more parts of aplant, which can include above ground and/or below ground (harvestable)plant parts. In particular, such harvestable parts are seeds, andperformance of the methods of the disclosure results in plants withincreased yield and in particular increased seed yield relative to theseed yield of suitable control plants. The term “yield” of a plant canrelate to vegetative biomass (root and/or shoot biomass), toreproductive organs, and/or to propagules (such as seeds) of that plant.

In an embodiment, “alfalfa yield” can also be measured in forage yield,the amount of above ground biomass at harvest, Factors leadingcontributing to increased biomass include increased vegetative growth,branches, nodes and internodes, leaf area, and leaf area index.

In another embodiment, “canola yield” can also be measured in podnumber, number of pods per plant, number of pods per node, number ofinternodes, incidence of pod shatter, seeds per silique, seed weight persilique, improved seed, oil, or protein composition.

Additionally, “corn or maize yield” can also be measured as productionof shelled corn kernels per unit of production area, ears per acre,number of kernel rows per ear, weight per kernel, ear number, fresh ordry ear biomass (weight), kernel rows per ear and kernels per row.

In yet another embodiment, “cotton yield” can be measured as bolls perplant, size of bolls, fiber quality, seed cotton yield in g/plant, seedcotton yield in lb/acre, lint yield in lb/acre, and number of bales.

Specific embodiments for “rice yield” can also include panicles perhill, grain per hill, and filled grains per panicle.

Still further embodiment for “soybean yield” can also include pods perplant, pods per acre, seeds per plant, seeds per pod, weight per seed,weight per pod, pods per node, number of nodes, and the number ofinternodes per plant.

In still further embodiments, “sugarcane yield” can be measured as caneyield (tons per acre; kg/hectare), total recoverable sugar (pounds perton), and sugar yield (tons/acre).

In yet still further embodiments, “wheat yield” can include: cereal perunit area, grain number, grain weight, grain size, grains per head,seeds per head, seeds per plant, heads per acre, number of viabletillers per plant, composition of seed (for example, carbohydrates,starch, oil, and protein) and characteristics of seed fill.

The terms “yield”, “seed yield” are defined above for a number of corecrops. The terms “increased”, “improved”, “enhanced” are interchangeableand are defined herein.

The present disclosure also provides a method for the production ofplants having increased yield. Performance of the method gives plantshaving increased yield. “Increased yield” can manifest as one or more ofthe following: (i) increased plant biomass (weight) of one or more partsof a plant, particularly aboveground (harvestable) parts, of a plant,increased root biomass (increased number of roots, increased rootthickness, increased root length) or increased biomass of any otherharvestable part; (ii) increased early vigor, defined herein as animproved seedling aboveground area approximately three weekspost-germination. “Early vigor” refers to active healthy plant growthespecially during early stages of plant growth, and can result fromincreased plant fitness due to, for example, the plants being betteradapted to their environment (for example, optimizing the use of energyresources, uptake of nutrients and partitioning carbon allocationbetween shoot and root). Early vigor in corn, for example, is acombination of the ability of corn seeds to germinate and emerge afterplanting and the ability of the young corn plants to grow and developafter emergence. Plants having early vigor also show increased seedlingsurvival and better establishment of the crop, which often results inhighly uniform fields with the majority of the plants reaching thevarious stages of development at substantially the same time, whichoften results in increased yield. Therefore early vigor can bedetermined by measuring various factors, such as kernel weight,percentage germination, percentage emergence, seedling growth, seedlingheight, root length, root and shoot biomass, canopy size and color andothers; (iii) increased total seed yield, which includes an increase inseed biomass (seed weight) and which can be an increase in the seedweight per plant or on an individual seed basis; increased number ofpanicles per plant; increased pods, increased number of nodes, increasednumber of flowers (“florets”) per panicle/plant; increased seed fillrate; increased number of filled seeds; increased seed size (length,width, area, perimeter), which can also influence the composition ofseeds; increased seed volume, which can also influence the compositionof seeds. Increased yield can also result in modified architecture, orcan occur because of modified plant architecture; (iv) increased harvestindex, which is expressed as a ratio of the yield of harvestable parts,such as seeds, over the total biomass; and (v) increased kernel weight,which is extrapolated from the number of filled seeds counted and theirtotal weight. An increased kernel weight can result from an increasedseed size and/or seed weight, an increase in embryo size, endospermsize, aleurone and/or scutellum, or other parts of the seed.

In one embodiment, increased yield can be increased seed yield, and isselected from one of the following: (i) increased seed weight; (ii)increased number of filled seeds; and (iii) increased harvest index.

The disclosure also extends to harvestable parts of a plant such as, butnot limited to, seeds, leaves, fruits, flowers, bolls, stems, rhizomes,tubers and bulbs. The disclosure furthermore relates to products derivedfrom a harvestable part of such a plant, such as dry pellets, powders,oil, fat and fatty acids, starch or proteins.

The present disclosure provides a method for increasing “yield” of aplant or “broad acre yield” of a plant or plant part defined as theharvestable plant parts per unit area, for example seeds, or weight ofseeds, per acre, pounds per acre, bushels per acre, tones per acre, tonsper acre, kilo per hectare.

This disclosure further provides a method of increasing yield in a plantby producing a plant comprising a polynucleic acid sequence encoding apolypeptide of this disclosure where the plant can be crossed withitself, a second plant from the same plant line, a wild type plant, or aplant from a different line of plants to produce a seed. The seed of theresultant plant can be harvested from fertile plants and be used to growprogeny generations of plants) of this disclosure. In addition to directtransformation of a plant with a recombinant DNA, transgenic plants canbe prepared by crossing a first plant having a recombinant DNA with asecond plant lacking the DNA. For example, recombinant DNA can beintroduced into a first, plant line that is amenable to transformationto produce a transgenic plant which can be crossed with a second plantline to introgress the recombinant DNA into the second plant line. Atransgenic plant with a recombinant DNA having the polynucleotide ofthis disclosure provides the enhanced trait of increased yield comparedto a control plant. Genetic markers associated with recombinant DNA canproduce transgenic progeny that is homozygous for the desiredrecombinant DNA, Progeny plants carrying DNA for both parental traitscan be back crossed into a parent line multiple times, for exampleusually 6 to 8 generations, to produce a progeny plant withsubstantially the same genotype as the one original transgenic parentalline but having the recombinant DNA of the other transgenic parentalline. The term “progeny” denotes the offspring of any generation of aparent plant prepared by the methods of this disclosure containing therecombinant polynucleotides as described herein.

As used herein “nitrogen use efficiency” refers to the processes whichlead to an increase in the plant's yield, biomass, vigor, and growthrate per nitrogen unit applied. The processes can include the uptake,assimilation, accumulation, signaling, sensing, translocation (withinthe plant) and use of nitrogen by the plant.

As used herein “nitrogen limiting conditions” refers to growthconditions or environments that provide less than optimal amounts ofnitrogen needed for adequate or successful plant metabolism, growth,reproductive success and/or viability.

As used herein the “increased nitrogen stress tolerance” refers to theability of plants to grow, develop, or yield normally, or grow, develop,or yield faster or better when subjected to less than optimal amounts ofavailable/applied nitrogen, or under nitrogen limiting conditions.

As used herein “increased nitrogen use efficiency” refers to the abilityof plants to grow, develop, or yield faster or better than normal whensubjected to the same amount of available/applied nitrogen as undernormal or standard conditions; ability of plants to grow, develop, oryield normally, or grow, develop, or yield faster or better whensubjected to less than optimal amounts of available/applied nitrogen, orunder nitrogen limiting conditions.

Increased plant nitrogen use efficiency can be translated in the fieldinto either harvesting similar quantities of yield, while supplying lessnitrogen, or increased yield gained by supplying optimal/sufficientamounts of nitrogen. The increased nitrogen use efficiency can improveplant nitrogen stress tolerance, and can also improve crop quality andbiochemical constituents of the seed such as protein yield and oilyield. The terms “increased nitrogen use efficiency”, “enhanced nitrogenuse efficiency”, and “nitrogen stress tolerance” are usedinter-changeably in the present disclosure to refer to plants withimproved productivity under nitrogen limiting conditions.

As used herein “water use efficiency” refers to the amount of carbondioxide assimilated by leaves per unit of water vapor transpired. Itconstitutes one of the most important traits controlling plantproductivity in dry environments. “Drought tolerance” refers to thedegree to which a plant is adapted to and or drought conditions. Thephysiological responses of plants to a deficit of water include leafwilting, a reduction in leaf area, leaf abscission, and the stimulationof root growth by directing nutrients to the underground parts of theplants. Plants are more susceptible to drought during flowering and seeddevelopment (the reproductive stages), as plant's resources are deviatedto support root growth. In addition, abscisic acid (ABA), a plant stresshormone, induces the closure of leaf stomata (microscopic pores involvedin gas exchange), thereby reducing water loss through transpiration, anddecreasing the rate of photosynthesis. These responses improve thewater-use efficiency of the plant on the short term. The terms“increased water use efficiency”, “enhanced water use efficiency” and“increased drought tolerance” are used inter-changeably in the presentdisclosure to refer to plants with improved productivity underwater-limiting conditions.

As used herein “increased water use efficiency” refers to the ability ofplants to grow, develop, or yield faster or better than normal whensubjected to the same amount of available/applied water as under normalor standard conditions; ability of plants to grow, develop, or yieldnormally, or grow, develop, or yield faster or better when subjected toreduced amounts of available/applied water (water input) or underconditions of water stress or water deficit stress.

As used herein “increased drought tolerance” refers to the ability ofplants to grow, develop, or yield normally, or grow, develop, or yieldfaster or better than normal when subjected to reduced amounts ofavailable/applied water and/or under conditions of acute or chronicdrought; ability of plants to grow, develop, or yield normally whensubjected to reduced amounts of available/applied water (water input) orunder conditions of water deficit stress or under conditions of acute orchronic drought.

As used herein “drought stress” refers to a period of dryness (acute orchronic/prolonged) that results in water deficit and subjects plants tostress and/or damage to plant tissues and/or negatively affectsgrain/crop yield; a period of dryness (acute or chronic/prolonged) thatresults in water deficit and/or higher temperatures and subjects plantsto stress and/or damage to plant tissues and/or negatively affectsgrain/crop yield.

As used herein “water deficit” refers to the conditions or environmentsthat provide less than optimal amounts of water needed foradequate/successful growth and development of plants.

As used herein “water stress” refers to the conditions or environmentsthat provide improper (either less/insufficient or more/excessive)amounts of water than that needed for adequate/successful growth anddevelopment of plants/crops thereby subjecting the plants to stressand/or damage to plant tissues and/or negatively affecting grain/cropyield.

As used herein “water deficit stress” refers to the conditions orenvironments that provide less/insufficient amounts of water than thatneeded for adequate/successful growth and development of plants/cropsthereby subjecting the plants to stress and/or damage to plant tissuesand/or negatively affecting grain yield.

As used herein a “polynucleotide” is a nucleic acid molecule comprisinga plurality of polymerized nucleotides. A polynucleotide may be referredto as a nucleic acid, oligonucleotide, nucleotide, or any fragmentthereof. In many instances, a polynucleotide encodes a polypeptide (orprotein) or a domain or fragment thereof. Additionally, a polynucleotidecan comprise a promoter, an intron, an enhancer region, apolyadenylation site, a translation initiation site, 5° or 3′untranslated regions, a reporter gene, a selectable marker, a scorablemarker, or the like. A polynucleotide can be single-stranded ordouble-stranded DNA or RNA. A polynucleotide optionally comprisesmodified, bases or a modified backbone. A polynucleotide can be, forexample, genomic DNA or RNA, a transcript (such as an mRNA), a cDNA, aPCR product, a cloned DNA, a synthetic DNA or RNA, or the like. Apolynucleotide can be combined with carbohydrate(s), lipid(s),protein(s), or other materials to perform a particular activity such astransformation or form a composition such as a peptide nucleic acid(PNA). A polynucleotide can comprise a sequence in either sense orantisense orientations. “Oligonucleotide” is substantially equivalent tothe terms amplimer, primer, oligomer, element, target, and probe and ispreferably single-stranded.

As used herein a “recombinant polynucleotide” or “recombinant DNA” is apolynucleotide that is not in its native state, for example, apolynucleotide comprises a series of nucleotides (represented as anucleotide sequence) not found in nature, or a polynucleotide is in acontext other than that in which it is naturally found; for example,separated from polynucleotides with which it typically is in proximityin nature, or adjacent (or contiguous with) polynucleotides with whichit typically is not in proximity. The “recombinant polynucleotide” or“recombinant DNA” refers to polynucleotide or DNA which has beengenetically engineered and constructed outside of a cell including DNAcontaining naturally occurring DNA or cDNA or synthetic DNA. Forexample, the polynucleotide at issue can be cloned into a vector, orotherwise recombined with one or more additional nucleic acids.

As used herein a “polypeptide” comprises a plurality of consecutivepolymerized amino acid residues for example, at least about 15consecutive polymerized amino acid residues. In many instances, apolypeptide comprises a series of polymerized amino acid residues thatis a transcriptional regulator or a domain or portion or fragmentthereof. Additionally, the polypeptide can comprise: (i) a localizationdomain; (ii) an activation domain; (iii) a repression domain; (iv) anoligomerization domain; (v) a protein-protein interaction domain; (vi) aDNA-binding domain; or the like. The polypeptide optionally comprisesmodified amino acid residues, naturally occurring amino acid residuesnot encoded by a codon, non-naturally occurring amino acid residues.

As used herein “protein” refers to a series of amino acids,oligopeptide, peptide, polypeptide or portions thereof whether naturallyoccurring or synthetic.

Recombinant DNA constructs are assembled using methods known to personsof ordinary skill in the art and typically comprise a promoter operablylinked to DNA, the expression of which provides the enhanced agronomictrait. Other construct components can include additional regulatoryelements, such as 5′ leaders and introns for enhancing transcription, 3′untranslated regions (such as polyadenylation signals and sites), andDNA for transit or targeting or signal peptides.

As used herein a “recombinant polypeptide” is a polypeptide produced bytranslation of a recombinant polynucleotide.

A “synthetic polypeptide” is a polypeptide created by consecutivepolymerization of isolated amino acid residues using methods known inthe art.

An “isolated polypeptide”, whether a naturally occurring or arecombinant polypeptide, is more enriched in (or out of) a cell than thepolypeptide in its natural state in a wild-type cell, for example, morethan about 5% enriched, more than about 10% enriched, or more than about20%, or more than about 50%, or more, enriched, for example,alternatively denoted: 105%, 110%, 120%, 150% or more, enriched relativeto wild type standardized at 100%. Such an enrichment is not the resultof a natural response of a wild-type plant. Alternatively, oradditionally, the isolated polypeptide is separated from other cellularcomponents with which it is typically associated, for example, by any ofthe various protein purification methods.

A “DNA construct” as used in the present disclosure comprises at leastone expression cassette having a promoter operable in plant cells and apolynucleotide of the present disclosure encoding a protein or variantof a protein or fragment of a protein that is functionally defined tomaintain activity in transgenic host cells including plant cells, plantparts, explants and plants. DNA constructs are made that contain variousgenetic elements necessary for the expression of noncoding and codingpolynucleotides in plants. Promoters, leaders, enhancers, introns,transit or targeting or signal peptides, 3′ transcriptional terminationregions are genetic elements that can be operably linked in a DNAconstruct.

Percent identity describes the extent to which polynucleotides orprotein segments are invariant in an alignment of sequences, for examplenucleotide sequences or amino acid sequences. An alignment of sequencesis created by manually aligning two sequences, for example, a statedsequence, as provided herein, as a reference, and another sequence, toproduce the highest number of matching elements, for example, individualnucleotides or amino acids, while allowing for the introduction of gapsinto either sequence. An “identity fraction” for a sequence aligned witha reference sequence is the number of matching elements, divided by thefull length of the reference sequence, not including gaps introduced bythe alignment process into the reference sequence. “Percent identity”(“% identity”) as used herein is the identity fraction times 100.

As used herein, a “functional fragment” refers to a portion of apolypeptide provided herein which retains full or partial molecular,physiological or biochemical function of the full length polypeptide. Afunctional fragment often contains the domain(s), such as Pfam domains,identified in the polypeptide provided in the sequence listing.

As used herein, a “homolog” or “homologues” means a protein in a groupof proteins that perform the same biological function, for example,proteins that belong to the same Pfam protein family and that provide acommon enhanced trait in transgenic plants of this disclosure. Homologsare expressed by homologous genes. With reference to homologous genes,homologs include orthologs, for example, genes expressed in differentspecies that evolved from a common ancestral genes by speciation andencode proteins retain the same function, but do not include paralogs,for example, genes that are related by duplication but have evolved toencode proteins with different functions. Homologous genes includenaturally occurring alleles and artificially-created variants.Degeneracy of the genetic code provides the possibility to substitute atleast one base of the protein encoding sequence of a gene with adifferent base without causing the amino acid sequence of thepolypeptide produced from the gene to be changed. When optimallyaligned, homolog proteins, or their corresponding nucleotide sequences,have typically at least about 60% identity, in some instances at leastabout 70%, at least about 75%, at least about bout 80%, at least about85%, at least about 90%, at least about bout 92%, at least about bout94%, at least about bout 95%, at least about 96%, at least about 97%, atleast about 98%, at least about 99%, and even at least about 99.5%identity over the full length of a protein or its correspondingnucleotide sequence identified as being associated with imparting anenhanced trait when expressed in plant cells. In one aspect of thedisclosure homolog proteins have an amino acid sequences that have atleast about 80%, at least about 85%, at least about 90%, at least about92%, at least about 94%, at least about 95%, at least about 96%, atleast about 97%, at least about 98%, at least about 99%, and at leastabout 99.5% identity to a consensus amino acid sequence of proteins andhomologs that can be built from sequences disclosed herein.

Homologs are inferred from sequence similarity, by comparison of proteinsequences, for example, manually or by use of a computer-based toolusing well-known sequence comparison algorithms such as BLAST and FASTA.A sequence search and local alignment program, for example, BLAST, canbe used to search query protein sequences of a base organism against adatabase of protein sequences of various organisms, to find similarsequences, and the summary Expectation value (E-value) can be used tomeasure the level of sequence similarity. Because a protein hit with thelowest E-value for a particular organism may not necessarily be anortholog or be the only ortholog, a reciprocal query is used to filterhit sequences with significant E-values for ortholog identification. Thereciprocal query entails search of the significant hits against adatabase of protein sequences of the base organism. A hit can beidentified as an ortholog, when the reciprocal query's best hit is thequery protein itself or a paralog of the query protein. With thereciprocal query process orthologs are further differentiated fromparalogs among all the homologs, which allows for the inference offunctional equivalence of genes. A further aspect, of the homologsencoded by DNA useful in the transgenic plants of the invention arethose proteins that differ from a disclosed protein as the result ofdeletion or insertion of one or more amino acids in a native sequence.

Other functional homolog proteins differ in one or more amino acids fromthose of a trait-improving protein disclosed herein as the result of oneor more of the well-known conservative amino acid substitutions, forexample, valine is a conservative substitute for alanine and threonineis a conservative substitute for serine. Conservative substitutions foran amino acid within the native sequence can be selected from othermembers of a class to which the naturally occurring amino acid belongs.Representative amino acids within these various classes include, but arenot limited to: (1) acidic (negatively charged) amino acids such asaspartic acid and glutamic acid; (2) basic (positively charged) aminoacids such as arginine, histidine, and lysine; (3) neutral polar aminoacids such as glycine, serine, threonine, cysteine, tyrosine,asparagine, and glutamine; and (4) neutral nonpolar (hydrophobic) aminoacids such as alanine, leucine, isoleucine, valine, proline,phenylalanine, tryptophan, and methionine. Conserved substitutes for anamino acid within a native protein or polypeptide can be selected fromother members of the group to which the naturally occurring amino acidbelongs. For example, a group of amino acids having aliphatic sidechains is glycine, alanine, valine, leucine, and isoleucine; a group ofamino acids having aliphatic-hydroxyl side chains is serine andthreonine; a group of amino acids having amide-containing side chains isasparagine and glutamine; a group of amino acids having aromatic sidechains is phenylalanine, tyrosine, and tryptophan; a group of aminoacids having basic side chains is lysine, arginine, and histidine; and agroup of amino acids having sulfur-containing side 30 chains is cysteineand methionine. Naturally conservative amino acids substitution groupsare: valine-leucine, valine-isoleucine, phenylalanine-tyrosine,lysine-arginine, alaninevaline, aspartic acid-glutamic acid, andasparagine-glutamine. A further aspect of the disclosure includesproteins that differ in one or more amino acids from those of adescribed protein sequence as the result of deletion or insertion of oneor more amino acids in a native sequence.

In general, the term “variant” refers to molecules with somedifferences, generated synthetically or naturally, in their nucleotideor amino acid sequences as compared to a reference (native)polynucleotides or polypeptides, respectively. These differences includesubstitutions, insertions, deletions or any desired combinations of suchchanges in a native polynucleotide or amino acid sequence.

With regard to polynucleotide variants, differences between presentlydisclosed polynucleotides and polynucleotide variants are limited sothat the nucleotide sequences of the former and the latter are similaroverall and, in many regions, identical. Due to the degeneracy of thegenetic code, differences between the former and the latter nucleotidesequences may be silent (for example, the amino acids encoded by thepolynucleotide are the same, and the variant polynucleotide sequenceencodes the same amino acid sequence as the presently disclosedpolynucleotide). Variant nucleotide sequences can encode different aminoacid sequences, in which case such nucleotide differences will result inamino acid substitutions, additions, deletions, insertions, truncationsor fusions with respect to the similarly disclosed polynucleotidesequences. These variations can result in polynucleotide variantsencoding polypeptides that share at least one functional characteristic.The degeneracy of the genetic code also dictates that many differentvariant polynucleotides can encode identical and/or substantiallysimilar polypeptides.

As used herein “gene” or “gene sequence” refers to the partial orcomplete coding sequence of a gene, its complement, and, its 5′ and/or3′ untranslated regions. A gene is also a functional unit ofinheritance, and in physical terms is a particular segment or sequenceof nucleotides along a molecule of DNA (or RNA, in the case of RNAviruses) involved in producing a polypeptide chain. The latter can besubjected to subsequent processing such as chemical modification orfolding to obtain a functional protein or polypeptide. By way ofexample, a transcriptional regulator gene encodes a transcriptionalregulator polypeptide, which can be functional or require processing tofunction as an initiator of transcription.

As used herein, the term “promoter” refers generally to a DNA moleculethat is involved in recognition and binding of RNA polymerase II andother proteins (trans-acting transcription factors) to initiatetranscription. A promoter can be initially isolated from the 5′untranslated region (5′ UTR) of a genomic copy of a gene. Alternately,promoters can be synthetically produced or manipulated DNA molecules.Promoters can also be chimeric, that is a promoter produced through thefusion of two or more heterologous DNA molecules. Plant promotersinclude promoter DNA obtained from plants, plant viruses, fungi andbacteria such as Agrobacterium and Bradyrhizobium bacteria.

Promoters which initiate transcription in all or most tissues of theplant are referred to as “constitutive” promoters. Promoters whichinitiate transcription during certain periods or stages of developmentare referred to as “developmental” promoters. Promoters whose expressionis enhanced in, certain tissues of the plant relative to other planttissues are referred to as “tissue enhanced” or “tissue preferred”promoters. Promoters which express within a specific tissue of theplant, with little or no expression in other plant tissues are referredto as “tissue specific” promoters. A promoter that expresses in acertain cell type of the plant, for example a microspore mother cell, isreferred to as a “cell type specific” promoter. An “inducible” promoteris a promoter in which transcription is initiated in response to anenvironmental stimulus such as cold, drought or light; or other stimulisuch as wounding or chemical application. Many physiological andbiochemical processes in plants exhibit endogenous rhythms with a periodof about 24 hours. A “diurnal promoter” is a promoter which exhibitsaltered expression profiles under the control of a circadian oscillator.Diurnal regulation is subject to environmental inputs such as light andtemperature and coordination by the circadian clock.

Sufficient expression in plant seed tissues is desired to affectimprovements in seed composition. Exemplary promoters for use for seedcomposition modification include promoters from seed genes such as napinas disclosed in U.S. Pat. No. 5,420,034, maize L3 oleosin as disclosedin U.S. Pat. No. 6,433,252, zein Z27 as disclosed by Russell et al.(1997) Transgenic Res, 6(2):157-166, globulin 1 as disclosed by Belangeret al (1991) Genetics 129:863-872, glutelin 1 as disclosed by Russell(1997) supra, and peroxiredoxin antioxidant (Peri) as disclosed by Stacyet al. (1996) Plant Mol Biol. 31(6):1205-1216.

As used herein, the term “leader” refers to a DNA molecule isolated fromthe untranslated 5′ region (5′ UTR) of a genomic copy of a gene and isdefined generally as a nucleotide segment between the transcriptionstart site (TSS) and the protein coding sequence start site.Alternately, leaders can be synthetically produced or manipulated DNAelements. A leader can be used as a 5′ regulatory element for modulatingexpression of an operably linked transcribable polynucleotide molecule.

As used herein, the term “intron” refers to a DNA molecule that can beisolated or identified from the genomic copy of a gene and can bedefined generally as a region spliced out during mRNA processing priorto translation. Alternately, an intron can be a synthetically producedor manipulated DNA element. An intron can contain enhancer elements thateffect the transcription of operably linked genes. An intron can be usedas a regulatory element for modulating expression of an operably linkedtranscribable polynucleotide molecule. A DNA construct can comprise anintron, and the intron may or may not be heterologous with respect tothe transcribable polynucleotide molecule.

As used herein, the term “enhancer” or “enhancer element” refers to acis-acting transcriptional regulatory element, a.k.a. cis-element, whichconfers an aspect of the overall expression pattern, but is usuallyinsufficient alone to drive transcription, of an operably linkedpolynucleotide. Unlike promoters, enhancer elements do not usuallyinclude a transcription start site (TSS) or TATA box or equivalentsequence. A promoter can naturally comprise one or more enhancerelements that affect the transcription of an operably linkedpolynucleotide. An isolated enhancer element can also be fused to apromoter to produce a chimeric promoter cis-element, which confers anaspect of the overall modulation of gene expression. A promoter orpromoter fragment can comprise one or more enhancer elements that effectthe transcription of operably linked genes. Many promoter enhancerelements are believed to bind DNA-binding proteins and/or affect DNAtopology, producing local conformations that selectively allow orrestrict access of RNA polymerase to the DNA template or that facilitateselective opening of the double helix at the site of transcriptionalinitiation. An enhancer element can function to bind transcriptionfactors that regulate transcription. Some enhancer elements bind morethan one transcription factor, and transcription factors can interactwith different affinities with more than one enhancer domain.

Expression cassettes of this disclosure can include a “transit peptide”or “targeting peptide” or “signal peptide” molecule located either 5′ or3′ to or within the gene(s). These terms generally refer to peptidemolecules that when linked to a protein of interest directs the proteinto a particular tissue, cell, subcellular location, or cell organelle.Examples include, but are not limited to, chloroplast transit peptides(CTPs), chloroplast targeting peptides, mitochondria targeting peptides,nuclear targeting signals, nuclear exporting signals, vacuolar targetingpeptides, vacuolar sorting peptides. For description of the use ofchloroplast transit peptides see U.S. Pat. Nos. 5,188,642 and 5,728,925.For description of the transit peptide region of an Arabidopsis EPSPSgene in the present disclosure, see Klee, H. J. et al (MGG (1987)210:437-442. Expression cassettes of this disclosure can also include anintron or introns. Expression cassettes of this disclosure can contain aDNA near the 3′ end of the cassette that acts as a signal to terminatetranscription from a heterologous nucleic acid and that directspolyadenylation of the resultant mRNA. These are commonly referred to as“3′-untranslated regions” or “3′-non-coding sequences” or “3′-UTRs”. The“3′ non-translated sequences” means DNA sequences located downstream ofa structural nucleotide sequence and include sequences encodingpolyadenylation and other regulatory signals capable of affecting mRNAprocessing or gene expression. The polyadenylation signal functions inplants to cause the addition of polyadenylate nucleotides to the 3′ endof the mRNA precursor. The polyadenylation signal can be derived from anatural gene, from a variety of plant genes, or from T-DNA. An exampleof a polyadenylation sequence is the nopaline synthase 3° sequence (nos3′; Fraley et al., Proc. Natl. Acad. Sci. USA 80: 4803-4807, 1983). Theuse of different 3′ non-translated sequences is exemplified byIngelbrecht et al., Plant Cell 1:671-680, 1989.

Expression cassettes of this disclosure can also contain one or moregenes that encode selectable markers and confer resistance to aselective agent such as an antibiotic or a herbicide. A number ofselectable marker genes are known in the art and can be used in thepresent disclosure: selectable marker genes conferring tolerance toantibiotics like kanamycin and paromomycin (nptII), hygromycin B (aphIV), spectinomycin (aadA), U.S. Patent Publication 2009/0138985A1 andgentamycin (aac3 and aacC4) or tolerance to herbicides like glyphosate(for example, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), U.S.Pat. Nos. 5,627,061; 5,633,435; 6,040,497; 5,094,945), sulfonylherbicides (for example, acetohydroxyacid synthase or acetolactatesynthase conferring tolerance to acetolactate synthase inhibitors suchas sulfonylurea, imidazolinone, triazolopyrimidine,pyrimidyloxybenzoates and phthalide (U.S. Pat. Nos. 6,225,105;5,767,366; 4,761,373; 5,633,437; 6,613,963; 5,013,659; 5,141,870;5,378,824; 5,605,011)), bialaphos or phosphinothricin or derivatives(e.g., phosphinothricin acetyltransferase (bar) tolerance tophosphinothricin or glufosinate (U.S. Pat. Nos. 5,646,024; 5,561,236;5,276,268; 5,637,489; 5,273,894); dicamba (dicamba monooxygenase, PatentApplication Publications US2003/0115626A1), or sethoxydim (modifiedacetyl-coenzyme A carboxylase for conferring tolerance tocyclohexanedione (sethoxydim)), and aryloxyphenoxypropionate (haloxyfop,U.S. Pat. No. 6,414,222).

Transformation vectors of this disclosure can contain one or more“expression cassettes”, each comprising a native or non-native plantpromoter operably linked to a polynucleotide sequence of interest, whichis operably linked to a 3′ UTR termination signal, for expression in anappropriate host cell. It also typically comprises sequences requiredfor proper translation of the polynucleotide or transgene. As usedherein, the term “transgene” refers to a polynucleotide moleculeartificially incorporated into a host cell's genome. Such a transgenecan be heterologous to the host cell. The term “transgenic plant” refersto a plant comprising such a transgene. The coding region usually codesfor a protein of interest but can also code fora functional RNA ofinterest, for example an antisense RNA, a nontranslated RNA, in thesense or antisense direction, a microRNA, a noncoding RNA, or asynthetic RNA used in either suppression or over expression of targetgene sequences. The expression cassette comprising the nucleotidesequence of interest can be chimeric, meaning that at least one of itscomponents is heterologous with respect to at least one of its othercomponents. As used herein the term “chimeric” refers to a DNA moleculethat is created from two or more genetically diverse sources, forexample a first, molecule from one gene or organism and a secondmolecule from another gene or organism.

Recombinant DNA constructs in this disclosure generally include a 3′element that typically contains a polyadenylation signal and site, Known3′ elements include those from Agrobacterium tumefaciens genes such asnos 3′, tml 3′, tmr 3′, tms 3′, ocs 3′, tr7 3′, for example disclosed inU.S. Pat. No. 6,090,627; 3′ elements from plant genes such as wheat(Triticum aesevitum) heat shock protein 17 (Hsp17 3′), a wheat ubiquitingene, a wheat fructose-1,6-biphosphatase gene, a rice glutelin gene, arice lactate dehydrogenase gene and a rice beta-tubulin gene, all ofwhich are disclosed in US Patent Application Publication 2002/0192813A1; and the pea (Pisum sativum) ribulose biphosphate carboxylase gene(rbs 3′), and 3′ elements from the genes within the host plant.

As used herein “operably linked” means the association of two or moreDNA fragments in a recombinant DNA, construct so that the function ofone, for example, protein-encoding DNA, is controlled by the other, forexample, a promoter.

As used herein “expressed” means produced, for example, a protein isexpressed in a plant cell when its cognate DNA is transcribed to mRNAthat is translated to the protein. An “expressed” protein can alsoinclude its truncated version (for example, N-terminal truncated,C-terminal truncated or internal truncated) as long as the truncatedversion maintains the same or similar functionality as the full lengthversion.

Transgenic plants can comprise a stack of one or more polynucleotidesdisclosed herein resulting in the production of multiple polypeptidesequences. Transgenic plants comprising stacks of polynucleotides can beobtained by either or both of traditional breeding methods or throughgenetic engineering methods. These methods include, but are not limitedto, crossing individual transgenic lines each comprising apolynucleotide of interest, transforming a transgenic plant comprising afirst gene disclosed herein with a second gene, and co-transformation ofgenes into a single plant cell. Co-transformation of genes can becarried out using single transformation vectors comprising multiplegenes or genes carried separately on multiple vectors.

Transgenic plants comprising or derived from plant cells of thisdisclosure transformed with recombinant DNA can be further enhanced withstacked traits, for example, a crop plant having an enhanced traitresulting from expression of DNA disclosed herein in combination withherbicide and/or pest resistance traits. For example, genes of thecurrent disclosure can be stacked with other traits of agronomicinterest, such as a trait providing herbicide resistance, or insectresistance, such as using a gene from Bacillus thuringensis to provideresistance against lepidopteran, coliopteran, homopteran, hemiopteran,and other insects, or improved quality traits such as improvednutritional value. Herbicides for which transgenic plant tolerance hasbeen demonstrated and the method of the present disclosure can beapplied include, but are not limited to, glyphosate, dicamba,glufosinate, sulfonylurea, bromoxynil and norflurazon herbicides.Polynucleotide molecules encoding proteins involved in herbicidetolerance are well-known in the art and include, but are not limited to,a polynucleotide molecule encoding 5-enolpyruvylshikimate-3-phosphatesynthase (EPSPS) disclosed in U.S. Pat. Nos. 5,094,945; 5,627,061;5,633,435 and 6,040,497 for imparting glyphosate tolerance;polynucleotide molecules encoding a glyphosate oxidoreductase (GOX)disclosed in U.S. Pat. No. 5,463,175 and a glyphosate-N-acetyltransferase (GAT) disclosed in US Patent Application Publication2003/0083480 A1 also for imparting glyphosate tolerance; dicambamonooxygenase disclosed in US Patent Application Publication2003/0135879 A1 for imparting dicamba tolerance; a polynucleotidemolecule encoding bromoxynil nitrilase (Bxn) disclosed in U.S. Pat. No.4,810,648 for imparting bromoxynil tolerance; a polynucleotide moleculeencoding phytoene desaturase (crtI) described in Misawa et al, (1993)Plant J. 4:833-840 and in Misawa et al, (1994) Plant J. 6:481-489 fornorflurazon tolerance; a polynucleotide molecule encodingacetohydroxyacid synthase (AHAS, aka ALS) described in Sathasiivan etal. (1990) Nucl. Acids Res. 18:2188-2193 for imparting tolerance tosulfonylurea herbicides; polynucleotide molecules known as bar genesdisclosed in DeBlock, et al. (1987) EMBO J. 6:2513-2519 for impartingglufosinate and bialaphos tolerance; polynucleotide molecules disclosedin US Patent Application Publication 2003/010609 A1 for impartingN-amino methyl phosphonic acid tolerance; polynucleotide moleculesdisclosed in U.S. Pat. No. 6,107,549 for impartinig pyridine herbicideresistance; molecules and methods for imparting tolerance to multipleherbicides such as glyphosate, atrazine, ALS inhibitors, isoxoflutoleand glufosinate herbicides are disclosed in U.S. Pat. No. 6,376,754 andUS Patent Application Publication 2002/0112260. Molecules and methodsfor imparting insect/nematode/virus resistance are disclosed in U.S.Pat. Nos. 5,250,515; 5,880,275; 6,506,599; 5,986,175 and US PatentApplication Publication 2003/0150017 A1.

Plant Cell Transformation Methods

Numerous methods for transforming chromosomes in a plant cell withrecombinant DNA are known in the art and are used in methods ofproducing a transgenic plant cell and plant. Two effective methods forsuch transformation are Agrobacterium-mediated transformation andmicroprojectile bombardment-mediated transformation. Microprojectilebombardment methods are illustrated in U.S. Pat. No. 5,015,580(soybean); U.S. Pat. No. 5,550,318 (corn); U.S. Pat. No. 5,538,880(corn); U.S. Pat. No. 5,914,451 (soybean); U.S. Pat. No. 6,160,208(corn); U.S. Pat. No. 6,399,861 (corn); U.S. Pat. No. 6,153,812 (wheat)and U.S. Pat. No. 6,365,807 (rice). Agrobacterium-mediatedtransformation methods are described in U.S. Pat. No. 5,159,135(cotton); U.S. Pat. No. 5,824,877 (soybean); U.S. Pat. No. 5,463,174(canola); U.S. Pat. No. 5,591,616 (corn); U.S. Pat. No. 5,846,797(cotton); U.S. Pat. No. 6,384,301 (soybean), U.S. Pat. No. 7,026,528(wheat) and U.S. Pat. No. 6,329,571 (rice), US Patent ApplicationPublication 2004/0087030 A1 (cotton), and US Patent ApplicationPublication 2001/0042257 A1 (sugar beet), all of which are incorporatedherein by reference in their entirety. Transformation of plant materialis practiced in tissue culture on nutrient media, for example a mixtureof nutrients that allow cells to grow in vitro. Recipient cell targetsinclude, but are not limited to, meristem cells, shoot tips, hypocotyls,calli, immature or mature embryos, and gametic cells such asmicrospores, pollen, sperm and egg cells. Callus can be initiated fromtissue sources including, but not limited to, immature or matureembryos, hypocotyls, seedling apical meristerns, microspores and thelike. Cells containing a transgenic nucleus are grown into transgenicplants.

In addition to direct transformation of a plant material with arecombinant DNA, a transgenic plant can be prepared by crossing a firstplant comprising a recombinant DNA with a second plant lacking therecombinant DNA. For example, recombinant DNA can be introduced into afirst plant line that is amenable to transformation, which can becrossed with a second plant line to introgress the recombinant DNA intothe second plant line. A transgenic plant with recombinant DNA providingan enhanced trait, for example, enhanced yield, can be crossed with atransgenic plant line having other recombinant DNA that confers anothertrait, for example herbicide resistance or pest resistance, to produceprogeny plants having recombinant DNA that confers both traits.Typically, in such breeding for combining traits the transgenic plantdonating the additional trait is a male line and the transgenic plantcarrying the base traits is the female line. The progeny of this crosswill segregate such that some of the plants will carry the DNA for bothparental traits and some will carry DNA for one parental trait; suchplants can be identified by markers associated with parental recombinantDNA, for example, marker identification by analysis for recombinant DNAor in the case where a selectable marker is linked to the recombinant,by application of the selecting agent such as a herbicide for use with aherbicide tolerance marker, or by selection for the enhanced trait.Progeny plants carrying DNA for both parental traits can be crossed backinto the female parent line multiple times, for example usually 6 to 8generations, to produce a progeny plant with substantially the samegenotype as the original transgenic parental line but for therecombinant DNA of the other transgenic parental line.

For transformation, DNA is typically introduced into only a smallpercentage of target plant cells in any one transformation experiment.Marker genes are used to provide an efficient system for identificationof those cells that are stably transformed by receiving and integratinga recombinant DNA molecule into their genomes. Preferred marker genesprovide selective markers which confer resistance to a selective agent,such as an antibiotic or a herbicide. Any of the herbicides to whichplants of this disclosure can be resistant is a agent for selectivemarkers. Potentially transformed cells are exposed to the selectiveagent. In the population of surviving cells are those cells where,generally, the resistance-conferring gene is integrated and expressed atsufficient levels to permit cell survival. Cells can be tested furtherto confirm stable integration of the exogenous DNA. Commonly usedselective marker genes include those conferring resistance toantibiotics such as kanamycin and paromomycin (nptII), hygromycin B (aphIV), spectinomycin (aadA) and gentamycin (aac3 and aacC4) or resistanceto herbicides such as glufosinate (bar or pat), dicamba (DMO) andglyphosate (aroA or EPSPS). Examples of such selectable markers areillustrated in U.S. Pat. Nos. 5,550,318; 5,633,435; 5,780,708 and6,118,047. Markers which provide an ability to visually screentransformants can also be employed, for example, a gene expressing acolored or fluorescent protein such as a luciferase or green fluorescentprotein (GFP) or a gene expressing a beta-glucuronidase or uidA gene(GUS) for which various chromogenic substrates are known.

Plant cells that survive exposure to a selective agent, or plant cellsthat have been scored positive in a screening assay, may be cultured invitro to regenerate plantlets. Developing plantlets regenerated fromtransformed plant cells can be transferred to plant growth mix, andhardened off, for example, in an environmentally controlled chamber atabout 85% relative humidity, 600 ppm CO₂, and 25-250 microeinsteins m⁻²s⁻¹ of light, prior to transfer to a greenhouse or growth chamber formaturation. Plants are regenerated from about 6 weeks to 10 months aftera transformant is identified, depending on the initial tissue, and plantspecies. Plants can be pollinated using conventional plant breedingmethods known to those of skill in the art to produce seeds, for exampleself-pollination is commonly used with transgenic corn. The regeneratedtransformed plant or its progeny seed or plants can be tested forexpression of the recombinant DNA and selected for the presence of anenhanced agronomic trait.

Transgenic Plants and Seeds

Transgenic plants derived from transgenic plant cells having atransgenic nucleus of this disclosure are grown to generate transgenicplants having an enhanced trait as compared to a control plant, andproduce transgenic seed and haploid pollen of this disclosure. Suchplants with enhanced traits are identified by selection of transformedplants or progeny seed for the enhanced trait. For efficiency aselection method is designed to evaluate multiple transgenic plants(events) comprising the recombinant DNA, for example multiple plantsfrom 2 to 20 or more transgenic events. Transgenic plants grown fromtransgenic seeds provided herein demonstrate improved agronomic traitsthat contribute to increased yield or other traits that provideincreased plant value, including, for example, improved seed quality. Ofparticular interest are plants having increased water use efficiency ordrought tolerance, enhanced high temperature or cold tolerance,increased yield, increased nitrogen, use efficiency.

Table 1 provides a list of protein-encoding DNA (“genes”) as recombinantDNA for production of transgenic plants with enhanced traits, theelements of Table 1 are described by reference to:

“PEP SEQ ID NO” which identifies an amino acid sequence.

“NUC SEQ ID NO” which identifies a DNA sequence.

“Gene ID” which refers to an arbitrary identifier.

“Protein Name” which is a common name for protein encoded by therecombinant DNA.

TABLE 1 NUC PEP SEQ ID NO SEQ ID NO Gene ID Protein Name 1 2 TRDXM1-1OsNAC6 protein. 3 4 TRDXM1-2 WRKY transcription factor. 5 6 TRDXM1-3ERF-like protein 7 8 TRDXM1-4 PREDICTED: E3 ubiquitin- protein ligaseATL6-like. 9 10 TRDXM1-5 Putative bHLH transcription factor. 11 12TRDXM1-6 Putative zinc finger transcription factor 13 14 TRDXM1-7Putative DNA repair protein rhp54. 15 16 TRDXM1-8 Transcription factor-like protein bZIP1. 17 18 TRDXM1-9 Stress-induced transcription factorNAC1. 19 20 TRDXM1-10 Zinc finger protein.Selection Methods for Transgenic Plants with Enhanced Traits

Within a population of transgenic plants each regenerated from a plantcell with recombinant DNA many plants that survive to fertile transgenicplants that produce seeds and progeny plants will not exhibit anenhanced agronomic trait. Selection from the population is necessary toidentify one or more transgenic plants with an enhanced trait.Transgenic plants having enhanced traits are selected from populationsof plants regenerated or derived from plant cells transformed asdescribed herein by evaluating the plants in a variety of assays todetect an enhanced trait, for example, increased water use efficiency ordrought tolerance, enhanced high temperature or cold tolerance,increased yield, increased nitrogen use efficiency, enhanced seedcomposition such as enhanced seed protein and enhanced seed oil. Theseassays can take many forms including, but not limited to, directscreening for the trait in a greenhouse or field trial or by screeningfor a surrogate trait. Such analyses can be directed to detectingchanges in the chemical composition, biomass, physiological property, ormorphology of the plant. Changes in chemical compositions such asnutritional composition of grain can be detected by analysis of the seedcomposition and content of protein, free amino acids, oil, free fattyacids, starch or tocopherols. Changes in chemical compositions can alsobe detected by analysis of contents in leaves, such as chlorophyll orcarotenoid contents. Changes in biomass characteristics can be evaluatedon greenhouse or field grown plants and can include plant height, stemdiameter, root and shoot dry weights, canopy size; and, for corn plants,ear length and diameter. Changes in physiological properties can beidentified by evaluating responses to stress conditions, for exampleassays using imposed stress conditions such as water deficit, nitrogendeficiency, cold growing conditions, pathogen or insect attack or lightdeficiency, or increased plant density. Changes in morphology can bemeasured by visual observation of tendency of a transformed plant toappear to be a normal plant as compared to changes toward bushy, taller,thicker, narrower leaves, striped leaves, knotted trait, chlorosis,albino, anthocyanin production, or altered tassels, ears or roots. Otherselection properties include days to pollen shed, days to silking, leafextension rate, chlorophyll content, leaf temperature, stand, seedlingvigor, internode length, plant height, leaf number, leaf area,tillering, brace roots, stay green or deleyed senescence, stalk lodging,root lodging, plant health, barreness/prolificacy, green snap, and pestresistance. In addition, phenotypic characteristics of harvested graincan be evaluated, including number of kernels per row on the ear, numberof rows of kernels on the ear, kernel abortion, kernel weight, kernelsize, kernel density and physical grain quality.

Assays for screening for a desired trait are readily designed by thosepracticing in the art. The following illustrates screening assays forcorn traits using hybrid corn plants. The assays can be adapted forscreening other plants such as canola, wheat, cotton and soybean eitheras hybrids or inbreds.

Transgenic corn plants having increased nitrogen use efficiency can beidentified by screening transgenic plants in the field under the sameand sufficient amount of nitrogen supply as compared to control plants,where such plants provide higher yield as compared to control plants.Transgenic corn plants having increased nitrogen use efficiency can alsobe identified by screening transgenic plants in the field under reducedamount of nitrogen supply as compared to control plants, where suchplants provide the same or similar yield as compared to control plants.

Transgenic corn plants having increased yield are identified byscreening using progenies of the transgenic plants over multiplelocations for several years with plants grown under optimal productionmanagement practices and maximum weed and pest control. Selectionmethods can be applied in multiple and diverse geographic locations, forexample up to 16 or more locations, over one or more planting seasons,for example at least two planting seasons, to statistically distinguishyield improvement from natural environmental effects.

Transgenic corn plants having increased water use efficiency or droughttolerance are identified by screening plants in an assay where water iswithheld fora period to induce stress followed by watering to revive theplants. For example, a selection process imposes 3 drought/re-watercycles on plants over a total period of 15 days after an initial stressfree growth period of 11 days. Each cycle consists of 5 days, with nowater being applied for the first four days and a water quenching on the5th day of the cycle. The primary phenotypes analyzed by the selectionmethod are the changes in plant growth rate as determined by height andbiomass during a vegetative drought treatment.

Transgenic cotton plants with increased yield and increased water useefficiency are identified by growing under variable water conditions.Specific conditions for cotton include growing a first set of transgenicand control plants under “wet” conditions, for example irrigated in therange of 85 to 100 percent of evapotranspiration to provide leaf waterpotential of −14 to −18 bars, and growing a second set of transgenic andcontrol plants under “dry” conditions, for example irrigated in therange of 40 to 60 percent of evapotranspiration to provide a leaf waterpotential of −21 to −25 bars. Pest control, such as weed and insectcontrol is applied equally to both wet and dry treatments as needed.Data gathered during the trial includes weather records throughout thegrowing season including detailed records of rainfall; soilcharacterization information; any herbicide or insecticide applications;any gross agronomic differences observed such as leaf morphology,branching habit, leaf color, time to flowering, and fruiting pattern;plant height at various points during the trial; stand density; node andfruit number including node above white flower and node above crack bollmeasurements; and visual wilt scoring. Cotton boll samples are taken andanalyzed for lint fraction and fiber quality. The cotton is harvested atthe normal harvest timeframe for the trial area. Increased water useefficiency is indicated by increased yield, improved relative watercontent, enhanced leaf water potential, increased biomass, enhanced leafextension rates, and improved fiber parameters.

Although the plant cells and methods of this disclosure can be appliedto any plant cell, plant, seed or pollen, for example, any fruit,vegetable, grass, tree or ornamental plant, the various aspects of thedisclosure are applied to corn, soybean, cotton, canola, rice, barley,oat, wheat, turf grass, alfalfa, sugar beet, sunflower, quinoa and sugarcane plants.

Example 1. Corn Transformation

This example illustrates transformation methods in producing atransgenic corn plant cell, seed, and plant having altered phenotypes asshown in Tables 3-5, or an enhanced trait, for example, increased wateruse efficiency or drought tolerance, increased yield, increased nitrogenuse efficiency.

For Agrobacterium-mediated transformation of corn embryo cells cornplants were grown in the greenhouse and ears were harvested when theembryos were 1.5 to 2.0 mm in length. Ears were surface-sterilized byspraying or soaking the ears in 80% ethanol, followed by air drying.Immature embryos were isolated from individual kernels onsurface-sterilized ears. Shortly after excision, immature maize embryoswere inoculated with overnight grown Agrobacterium cells, and incubatedat room temperature with Agrobacterium for 5-20 minutes. Inoculatedimmature embryos were then co-cultured with Agrobacterium for 1 to 3days at 23° C. in the dark. Co-cultured embryos were transferred toselection media and cultured for approximately two weeks to allowembryogenic callus to develop. Embryogenic calli were transferred toculture medium containing glyphosate and subcultured at about two weekintervals. Transformed plant cells were recovered 6 to 8 weeks afterinitiation of selection.

For Agrobacterium-mediated transformation of maize callus immatureembryos are cultured for approximately 8-21 days after excision to allowcallus to develop. Callus is then incubated for about 30 minutes at roomtemperature with the Agrobacterium suspension, followed by removal ofthe liquid by aspiration. The callus and Agrobacterium are co-culturedwithout selection for 3-6 days followed by selection on paromomycin forapproximately 6 weeks, with biweekly transfers to fresh media.Paromomycin resistant calli are identified about 6-8 weeks afterinitiation of selection.

To regenerate transgenic corn plants individual transgenic calliresulting from transformation and selection were placed on media toinitiate shoot and root development into plantlets. Plantlets weretransferred to potting soil for initial growth in a growth chamber at26° C. followed by a mist bench before transplanting to 5 inch potswhere plants were grown to maturity. The regenerated plants wereself-fertilized and seeds were harvested for use in one or more methodsto select seeds, seedlings or progeny second generation transgenicplants (R2 plants) or hybrids, for example, by selecting transgenicplants exhibiting an enhanced trait as compared to a control plant.

The above process can be repeated to produce multiple events oftransgenic corn plants from cells that were transformed with recombinantDNA from the genes identified in Table 1. Progeny transgenic plants andseeds of the transformed plants were screened for the presence andsingle copy of the inserted gene, and for increased water useefficiency, increased yield, increased nitrogen use efficiency, andaltered phenotypes as shown in Tables 3-5. From each group of multipleevents of transgenic plants with a specific recombinant DNA from Table 1the event(s) that showed increased yield, increased water useefficiency, increased nitrogen use efficiency, and altered phenotypeswas (were) identified.

Example 2. Soybean Transformation

This example illustrates plant transformation in producing a transgenicsoybean plant cell, plant, and seed having an enhanced trait, forexample increased water use efficiency, increased yield, and increasednitrogen use efficiency.

For Agrobacterium mediated transformation, soybean seeds were imbibedovernight and the meristem explants excised. Soybean explants were mixedwith induced Agrobacterium cells containing plasmid DNA with the gene ofinterest cassette and a plant selectable marker cassette no later than14 hours from the time of initiation of seed imbibition, and woundedusing sonication. Following wounding, explants were placed in co-culturefor 2-5 days at which point they were transferred to selection media toallow selection and growth of transgenic shoots. Resistant shoots wereharvested in approximately 6-8 weeks and placed into selective rootingmedia for 2-3 weeks. Shoots producing roots were transferred to thegreenhouse and potted in soil. Shoots that remained healthy onselection, but did not produce roots were transferred to non-selectiverooting media for an additional two weeks. Roots from any shoots thatproduced roots off selection were tested for expression of the plantselectable marker before they were transferred to the greenhouse andpotted in soil.

The above process can be repeated to produce multiple events oftransgenic soybean plants from cells that were transformed withrecombinant DNA from the genes identified in Table 1. Progeny transgenicplants and seed of the transformed plant cells were screened for thepresence and single copy of the inserted gene, and for increased wateruse efficiency, increased yield, and increased nitrogen use efficiency.

Example 3. Cotton Transformation

This example illustrates plant transformation in producing a transgeniccotton plant cell, plant, and seed having an enhanced trait, for exampleincreased water use efficiency, increased yield, and increased nitrogenuse efficiency.

Transgenic cotton plants containing each recombinant DNA from the genesidentified in Table 1 were obtained by transforming cotton cells usingAgrobacterium-mediated transformation as described in U.S. Pat. Nos.7,790,460 and 7,947,869.

Progeny transgenic plants and seed of the transformed plant cells werescreened for the presence and single copy of the inserted gene, and forincreased water use efficiency, increased yield and increased nitrogenuse efficiency. From each group of multiple events of transgenic plantswith a specific recombinant DNA from Table 1 the event(s) that showedincreased yield, increased water use efficiency, and increased nitrogenuse efficiency was (were) identified.

Example 4. Canola Transformation

This example illustrates plant transformation in producing thetransgenic canola plants of this disclosure and the production andidentification of transgenic seed for transgenic canola having increasedwater use efficiency, increased yield, and increased nitrogen useefficiency.

Tissues from in vitro grown canola seedlings were prepared andinoculated with overnight-grown Agrobacterium cells containing plasmidDNA with a gene of interest cassette and a plant selectable markercassette. Following co-cultivation with Agrobacterium, the infectedtissues were allowed to grow on selection to promote growth oftransgenic shoots, followed by growth of roots from the transgenicshoots. The selected plantlets were then transferred to the greenhouseand potted in soil. Molecular characterizations were performed toconfirm the presence of the gene of interest, and its expression intransgenic plants and progenies. Progeny transgenic plants were selectedfrom a population of transgenic canola events under specified growingconditions and were compared with control canola plants.

The above process can be repeated to produce multiple events oftransgenic canola plants from cells that were transformed withrecombinant DNA from the genes identified in Table 1. Progeny transgenicplants and seed of the transformed plant cells were screened for thepresence and single copy of the inserted gene, and for increased wateruse efficiency, increased yield, and increased nitrogen use efficiency.From each group of multiple events of transgenic plants with a specificrecombinant DNA from Table 1 the event(s) that showed increased yield,increased water use efficiency, increased nitrogen use efficiency andaltered phenotypes was (were) identified.

Example 5. Identification of Altered Phenotypes in Automated Greenhouse

This example illustrates screening and identification of transgenicplants for altered phenotypes in an automated greenhouse (AGH). Theapparatus and the methods for automated phenotypic screening of plantsare disclosed in US Patent Publication No. US20110135161 (filed on Nov.10, 2010), which is incorporated by reference herein in its entirety.

Screening and Identification of Transgenic Corn Plants for AlteredPhenotypes.

Corn plants were tested in 3 screens in AGH under different conditionsincluding non-stress, nitrogen deficit and water deficit stressconditions. All screens began with a non-stress condition during day 0-5germination phase, after which the plants were grown for 22 days underscreen specific conditions as shown in Table 2.

Water deficit is defined as a specific Volumetric Water Content (V WC)that is lower than the VWC of non-stress plant. For example, anon-stressed plant might be maintained at 55% VWC and water-deficitassay might be defined around 30% VWC as shown in Table 2. Data werecollected using visible light and hyperspectral imaging as well asdirect measurement of pot weight and amount of water and nutrientapplied to individual plants on a daily basis.

Eight parameters were measured for each screen. The visible light colorimaging based measurements are: biomass, canopy area and plant height.Biomass (B) is defined as estimated shoot fresh weight (g) of the plantobtained from images acquired from multiple angles of view. Canopy Area(Can) is defined as area of leaf as seen in top-down image (mm²). PlantHeight (H) refers to the distance from the top of the pot to the highestpoint of the plant derived from side image (mm). Anthocyanin score,chlorophyll score and water content score are hyperspectral imagingbased parameters. Anthocyanin Score (An) is an estimate of anthocyaninin the leaf canopy obtained from a top-down hyperspectral image.Chlorophyll Score (Chl) is a measurement of chlorophyll in the leafcanopy obtained from a top-down hyperspectral image. Water Content Score(WC) is a measurement of water in the leaf canopy obtained from atop-down hyperspectral image. Water Use Efficiency (WUE) is derived fromthe grams of plant biomass per liter of water added. Water Applied (WA)is a direct measurement of water added to a pot (pot with no hole)during the course of an experiment.

These physiological screen runs were set up so that tested transgeniclines were compared to a control line. The collected data were analyzedagainst the control using % delta and certain p-value cutoff. Tables 3-5are summaries of transgenic corn plants comprising the disclosedrecombinant DNA molecules with altered phenotypes under non stress,nitrogen deficit, and water deficit conditions, respectively.

“+” denotes an increase in the tested parameter at p≤0.1; whereas “−”denotes a decrease in the tested parameter at p≤0.1. The numbers inparenthesis show penetrance of the altered phenotypes, where thedenominators represent total number of transgenic events tested for agiven parameter in a specific screen, and the numerators represent thenumber of events showing a particular altered phenotype. For example, 7transgenic plants were screened, for anthocyanin score in the non-stressscreen for TRDXM1-18 and 2 of the 7 tested showed increased anthocyaninat p≤0.1.

TABLE 2 Description of the 3 screens for corn plants. Germination Screenspecific phase phase Screen Description (5 days) (22 days) Non-stresswell watered 55% VWC 55% VWC sufficient nitrogen water 8 mM nitrogenWater deficit limited watered 55% VWC 30% VWC sufficient nitrogen water8 mM nitrogen Nitrogen deficit well watered 55% VWC 55% VWC low nitrogenwater 2 mM nitrogen

TABLE 3 Summary of transgenic corn plants with altered phenotypes in AGHnon-stress screens. Non-Stress Gene_ID An B H WA WUE TRDXM1-6 + (2/5) −(2/5) − (2/5) TRDXM1-7 − (2/7) TRDXM1-8 + (2/7) + (2/7) TRDXM1-9 − (4/5)− (2/5)

TABLE 4 Summary of transgenic corn plants with altered phenotypes in AGHnitrogen-deficit screens Nitrogen Deficit Gene_ID B Can Chl H WA WUETRDXM1-3 − (4/5) − (2/5) − (2/5) − (4/5) − (2/5) − (3/5) (Construct-624)TRDXM1-4 + (2/3) + (2/3) TRDXM1-6 + (5/5) + (4/5) + (5/5) + (5/5) +(5/5) + (5/5) TRDXM1-7 + (2/7) + (3/5) + (2/7) TRDXM1-8 − (2/7) TRDXM1-9− (2/5) − (2/5) − (4/5) + (2/5)

TABLE 5 Summary of transgenic corn plants with altered phenotypes in AGHwater-deficit screens Water Deficit Gene_ID An Can H WA WC TRDXM1-3 +(2/5) (Construct-624) TRDXM1-4 + (2/3) + (2/3) TRDXM1-6 + (2/4) +(3/4) + (2/4) TRDXM1-7 + (2/7) TRDXM1-8 + (3/7) + (2/7) TRDXM1-9 +(2/5) + (2/5) + (3/5)

Example 6. Phenotypic Evaluation of Transgenic Plants for EnhancedNitrogen Use Efficiency

Corn Nitrogen field efficacy trials were conducted to identify genesthat can improve nitrogen use efficiency under nitrogen limitingconditions leading to increased yield performance as compared to nontransgenic controls. A yield increase in corn can be manifested as oneor more of the following: an increase in the number of ears per plant,an increase in the number of rows, number of kernels per row, kernelweight, thousand kernel weight, fresh or dry ear length/diameter/biomass(weight), increase in the seed filling rate (which is the number offilled seeds divided by the total number of seeds and multiplied by100), among others. For the Nitrogen field trial results shown in Table6, each field was planted under nitrogen limiting condition (60lbs/acre) and the corn ear weight or yield was compared to controlplants to measure the yield increases.

Table 6 provides a list of protein encoding DNA or polynucleotidesequences (“genes”) for producing transgenic corn plant with increasednitrogen use efficiency as compared to a control plant. Polynucleotidesequences in constructs with at least one event showing significantyield or ear weight increase across multiple locations at p≤0.2 areincluded. The elements of Table 6 are described by reference to:

“SEQ ID NO: polynucleotide” which identifies a nucleotide sequence fromSEQ ID NO: 5, 7, 13, and 17.

“SEQ ID NO: polypeptide” which identifies an amino acid sequence fromSEQ ID NO: 6, 8, 14, and 18.

“Gene identifier” which refers to an arbitrary identifier.

“NUE results” refers to the sequence in a construct with at least oneevent showing significant yield increase at p≤0.2 across locations. Thefirst number refers to the number of events with significant yield orear weight increase, whereas the second number refers to the totalnumber of events tested for each sequence in a construct.

TABLE 6 Recombinant DNA for increased nitrogen use efficiency in cornSEQ ID NO: SEQ ID NO: Gene Nitrogen field trial polynucleotidepolypeptide Identifier Results 5 6 TRDXM1-3 0/5 2/8 2/5 (Construct-624)(Year-1: (Year-2: (Year-3: fresh ear yield) fresh ear weight) weight) 56 TRDXM1-3 1/5 (fresh (Construct-595) ear weight) 7 8 TRDXM1-4 1/5(yield) 13 14 TRDXM1-7 1/5 (yield) 17 18 TRDXM1-9 2/5 1/6 (Year-1:(Year-2: fresh ear yield) weight)

Example 7. Phenotypic Evaluation of Transgenic Plants for IncreasedYield

This example illustrates selection and identification of transgenicplants for increased yield, in both dicotyledonous and monocotyledonousplants with primary examples presented for corn and canola are presentedin Table 7 and 8 respectively. Polynucleotide sequences in constructswith at least one event that resulted in significant yield increaseacross locations at p≤0.2 are included.

Selection of Transgenic Plants with Enhanced Agronomic Trait(s):Increased Yield.

Effective selection of increased and/or enhanced yielding transgenicplants uses hybrid progenies of the transgenic plants for corn, cotton,and canola, or inbred progenies of transgenic plants for soybean plantsplant such as corn, cotton, canola, or inbred plant such as soy, canolaand cotton over multiple locations with plants grown under optimalproduction management practices. An exemplary target for improved yieldis a 2% to 10% increase in yield as compared to yield produced by plantsgrown from seed of a control plant. Selection methods can be applied inmultiple and diverse geographic locations, for example up to 16 or morelocations, over one or more planting seasons, for example at least twoplanting seasons, to statistically distinguish yield improvement fromnatural environmental effects.

Increased Yield in Corn

Table 7 provides a list of protein encoding DNA or polynucleotidesequences (“genes”) in the production of transgenic corn plant withincreased yield as compared to a control plant. The elements of Table 7are described by reference to:

“SEQ ID NO: polynucleotide” which identifies a nucleotide sequence fromSEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, and 19.

“SEQ ID NO: polypeptide” which identifies an amino acid sequence fromSEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, and 20.

“Gene identifier” which refers to an arbitrary identifier.

“Broad acre yield results” refers to the sequence in a construct with atleast one event showing significant yield increase at p≤0.2 acrosslocations. The first number refers to the number of events withsignificant yield increase, whereas the second number refers to thetotal number of events tested for each sequence in a construct. Asindicated in Table 7, gene TRDXM1-3 was tested in two constructs andsignificantly positive events were identified for both constructs.

TABLE 7 Recombinant DNA for increased nitrogen use efficiency in cornSEQ ID NO: SEQ ID NO: Gene Broad Acre Yield polynucleotide polypeptideIdentifier Results 3 4 TRDXM1-2 1/7 1/3 (Year-1) (Year-2) 5 6 TRDXM1-32/8 1/6 (Construct-624) (Year-1) (Year-2) 5 6 TRDXM1-3 2/8(Construct-595) 7 8 TRDXM1-4 0/6 1/3 2/3 (Year-1) (Year-2) (Year-3) 9 10TRDXM1-5 1/7 1/7 (Year-1) (Year-2) 11 12 TRDXM1-6 1/8 13 14 TRDXM1-7 2/74/6 1/6 (Year-1) (Year-2) (Year-3) 15 16 TRDXM1-8 4/8 17 18 TRDXM1-9 1/819 20 TRDXM1-10 1/8

Increased Yield in Canola

A yield increase in canola can be manifested as one or more of thefollowing: an increase in pod number, number of pods per plant, numberof pods per node, number of internodes, incidence of pod shatter, seedsper silique, seed weight per silique, improved seed, oil, or proteincomposition.

Table 8 provides a list of protein encoding DNA or polynucleotidesequences (“genes”) in the production of transgenic canola plant withincreased yield as compared to a control plant. The elements of Table 8are described by reference to:

“SEQ ID NO: polynucleotide” identifies a nucleotide sequence from SEQ IDNO: 1.

“SEQ ID NO: polypeptide” identifies an amino acid sequence from SEQ IDNO: 2.

“Gene identifier” which refers to an arbitrary identifier.

“Broad acre yield results” refers to the sequence in a construct with atleast one event showing significant yield increase at p≤0.2 acrosslocations. The first number refers to the number of events withsignificant yield increase, whereas the second number refers to thetotal number of events tested for each sequence in a construct.

TABLE 8 Canola Yield SEQ ID NO: SEQ ID NO: Gene Broad Acre Yieldpolynucleotide polypeptide Identifier Results 1 2 TRDXM1-1 1/8

Example 8. Homolog Identification

This example illustrates the identification of homologs of proteinsencoded by the DNA identified in Table 1 which were used to providetransgenic seed and plants having enhanced agronomic traits. From thesequences of the homolog proteins, corresponding homologous DNAsequences can be identified for preparing additional transgenic seedsand plants with enhanced agronomic traits.

An “All Protein Database” was constructed of known protein sequencesusing a proprietary sequence database and the National Center forBiotechnology Information (NCBI) non-redundant amino acid database(nr.aa). For each organism from which a polynucleotide sequence providedherein was obtained, an “Organism Protein Database” was constructed ofknown protein sequences of the organism; it is a subset of the AllProtein Database based on the NCBI taxonomy ID for the organism.

The All Protein Database was queried using amino acid sequences providedin Table 1 using NCBI “blastp” program with E-value cutoff of 1e-8. Upto 1000 top hits were kept, and separated by organism names. For eachorganism other than that of the query sequence, a list was kept for hitsfrom the query organism itself with a more significant E-value than thebest hit of the organism. The list contains likely duplicated genes ofthe polynucleotides provided herein, and is referred to as the CoreList. Another list was kept for all the hits from each organism, sortedby E-value, and referred to as the Hit List.

The Organism Protein Database was queried using polypeptide sequencesprovided in Table 1 using NCBI “blastp” program with E-value cutoff of1e-4. Up to 1000 top hits were kept. A BLAST searchable database wasconstructed based on these hits, and is referred to as “SubDB”. SubDB isqueried with each sequence in the Hit List using NCBI “blastp” programwith E-value cutoff of 1e-8. The hit with the best E-value was comparedwith the Core List from the corresponding organism. The hit is deemed alikely ortholog if it, belongs to the Core List, otherwise it is deemednot a likely ortholog and there is no further search of sequences in theHit List for the same organism. Homologs with at least 95% identity overthe 95% length of the polypeptide sequences provided, in Table 1 arereported below in Table 9 with the SEQ ID NO of the original querysequence and the identified homologs.

TABLE 9 Protein sequences and their homologs Peptide SEQ ID NO: HomologSEQ ID NOs 2 21, 22 4 23, 24, 25, 26 12 27 14 28 18 29

We claimed:
 1. A plant comprising a recombinant DNA molecule comprisinga polynucleotide encoding a polypeptide, wherein the nucleotide sequenceof the polynucleotide is selected from the group consisting of: a) anucleotide sequence set forth as SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15,17, or 19; b) a nucleotide sequence encoding a protein having the aminoacid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20-29; c)a nucleotide sequence with at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99% identity to SEQ ID NO: 1, 3, 5, 7, 9, 11, 13,15, 17, or 19; and d) a nucleotide sequence encoding a protein with atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%identity to SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20-29; whereinsaid plant has an enhanced trait as compared to a control plant.
 2. Theplant of claim 1, wherein said enhanced trait is selected from the groupconsisting of increased yield, increased nitrogen use efficiency, andincreased water use efficiency.
 3. The plant of claim 1, wherein saidplant is a monocot plant or is a member of the family Poaceae, wheatplant, maize plant, sweet corn plant, rice plant, wild rice plant,barley plant, rye, millet plant, sorghum plant, sugar cane plant,turfgrass plant, bamboo plant, oat plant, brome-grass plant, Miscanthusplant, pampas grass plant, switchgrass (Panicum) plant, and/or teosinteplant, or is a member of the family Alliaceae, onion plant, leek plant,garlic plant; or wherein the plant is a dicot plant or is a member ofthe family Amaranthaceae, spinach plant, quinoa plant, a member of thefamily Anacardiaceae, mango plant, a member of the family Asteraceae,sunflower plant, endive plant, lettuce plant, artichoke plant, a memberof the family Brassicaceae, Arabidopsis thaliana plant, rape plant,oilseed rape plant, broccoli plant, Brussels sprouts plant, cabbageplant, canola plant, cauliflower plant, kohlrabi plant, turnip plant,radish plant, a member of the family Bromeliaceae, pineapple plant, amember of the family Caricaceae, Papaya plant, a member of the familyChenopodiaceae, beet plant, a member of the family Curcurbitaceae, melonplant, cantaloupe plant, squash plant, watermelon plant, honeydew plant,cucumber plant, pumpkin plant, a member of the family Dioscoreaceae, yamplant, a member of the family Ericaceae, blueberry plant, a member ofthe family Euphorbiaceae, cassava plant, a member of the familyFabaceae, alfalfa plant, clover plant, peanut plant, a member of thefamily Grossulariaceae, currant plant, a member of the familyJuglandaceae, walnut plant, a member of the family Lamiaceae, mintplant, a member of the family Lauraceae, avocado plant, a member of thefamily Leguminosae, soybean plant, bean plant, pea plant, a member ofthe family Malvaceae, cotton plant, a member of the family Marantaceae,arrowroot plant, a member of the family Myrtaceae, guava plant,Eucalyptus plant, a member of the family Rosaceae, peach plant, appleplant, cherry plant, plum plant, pear plant, prune plant, blackberryplant, raspberry plant, strawberry plant, a member of the familyRubiaceae, coffee plant, a member of the family Rutaceae, citrus plant,orange plant, lemon plant, grapefruit plant, tangerine plant, a memberof the family Salicaceae, poplar plant, willow plant, a member of thefamily Solanaceae, potato plant, sweet potato plant, tomato plant,Capsicum plant, tobacco plant, tomatillo plant, eggplant plant, Atropabelladona plant, Datura stramonium plant, a member of the familyVitaceae, grape plant, a member of the family Umbelliferae, carrotplant, or a member of the family Musaceae, banana plant; or wherein theplant is a member of the family Pinaceae, cedar plant, fir plant,hemlock plant, larch plant, pine plant, or spruce plant.
 4. The plant ofclaim 1, wherein the recombinant DNA molecule further comprises apromoter that is operably linked to the polynucleotide encoding apolypeptide, wherein said promoter is selected from the group consistingof a constitutive, inducible, tissue specific, diurnally regulated,tissue enhanced, and cell specific promoter.
 5. The plant of claim 1,wherein said plant is a progeny, a propagule, or a field crop.
 6. Theplant of claim 5, wherein said field crop is selected from the groupconsisting of corn, soybean, cotton, canola, rice, barley, oat, wheat,turf grass, alfalfa, sugar beet, sunflower, quinoa and sugar cane. 7.The plant of claim 5, wherein said propagule is selected from the groupconsisting of a cell, pollen, ovule, flower, embryo, leaf, root, stem,shoot, meristem, grain and seed.
 8. A method for producing a plantcomprising: introducing into a plant cell a recombinant DNA moleculecomprising a polynucleotide encoding a polypeptide, wherein thenucleotide sequence of the polynucleotide is selected from the groupconsisting of: a) a nucleotide sequence set forth as SEQ ID NO: 1, 3, 5,7, 9, 11, 13, 15, 17, or 19; b) a nucleotide sequence encoding a proteinhaving the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16,18, or 20-29; c) a nucleotide sequence with at least 90%, at least 91%,at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99% identity to SEQ ID NO: 1, 3, 5, 7,9, 11, 13, 15, 17, or 19; and d) a nucleotide sequence encoding aprotein with at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99% identity to SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, or20-29; and growing a plant from said plant cell.
 9. The method of claim8, further comprising selecting a plant with an enhanced trait selectedfrom increased yield, increased nitrogen use efficiency, and increasedwater use efficiency as compared to a control plant.
 10. A method forincreasing yield, increasing nitrogen use efficiency, or increasingwater use efficiency in a plant comprising: crossing the plant of claim1 with itself, a second plant from the same plant line, a wild typeplant, or a second plant from a different line of plants to produce aseed; growing said seed to produce a plurality of progeny plants; andselecting a progeny plant with increased yield, increased nitrogen useefficiency, or increased water use efficiency.
 11. The plant of claim 1,wherein said plant has at least one phenotype selected from the groupconsisting of anthocyanin, biomass, canopy area, chlorophyll score,plant height, water applied, water content and water use efficiency thatis altered for said plant as compared to a control plant.